Effect Of Mutants Within Transmembrane Regions On Function Of ABC Transporter Pdr5P And Its Molecular Mechanism

Fungi cause significant morbidity and mortality in humans, especially among immunocompromised populations, such as patients with AIDS and recipients of tissue transplantation or chemotherapy. Prolonged usage of antifungal reagents can lead to drug resistance and treatment failure. Understanding mechanisms that underlie drug resistance by pathogenic microorganisms is thus vital for dealing with this emerging issue.The pathogenic Saccharomyces cerevisiae strain YJM789has many phenotypes that are relevant to its pathogenicity. In this study, segregant panel genotyped by the whole genome tiling array was used to perform genetic mapping to identify gene that is responsible for sensitivity difference to fluconazole between BY4741and YJM789. Combining with sequence analysis we found that dramatic sequence divergence in YJM789PDR5gene caused the organism to become hypersensitive to fluconazole.PDR5gene encodes an efflux pump that confers multidrug resistance by active efflux of a wide variety of structurally diverse toxic compounds. We tagged PDR5genes with GFP in both BY4741and YJM789. The results from fluorescence microscopy show that Pdr5p-GFP fusion localizes at plasma membrane in YJM789strain. Overexpression of YJM789PDR5fails to complement the drug-sensitive phenotype of pdr5deletion BY4741background strain. These results indicate that reduction of Pdr5p mediated drug resistance in YJM789is caused by impairment of Pdr5p function rather than mislocalization or down-regulated expression of Pdr5p.We compared the YJM789and BY4741for resistance to azole and polyene antifungal agents on drug agar plates and in liquid culture. Our results indicate that although the mutated PDR5gene in YJM789has lost the ability to facilitate growth in the presence of azoles, the same mutations conferred growth advantages to the organism on polyenes.To investigate the role of sequence divergence in the functional alterations of YJM789Pdr5p. A series of PDR5chimeric expression vectors were constructed by segmental replacement of divided BY4741PDR5fragments with their counterpart in YJM789PDR5. Functions of Pdr5p chimeras were evaluated by fluconazole and cycloheximide resistance assays. Their expression, subcellular localization, ATPase activity, and rhodamine6G efflux efficiency were also analyzed. Our results indicate that an alanine-to-methionine mutation at position1352located in the predicted short intracellular loop4which connects transmembrane helix10and transmembrane helix11caused severe decrease of drug export ability of Pdr5p. The degree of impairment is likely correlated to the size of the mutant residue. Our observation of the functional importance of A1352might encourage further studies on this region.